Reagent compositions for analytical testing

ABSTRACT

The present invention provides lyophilized reagent spheres comprising reagents suitable for analysis of biological samples, in particular analysis of blood samples in centrifugal analyzers. Also provided are diluents which are conveniently used in such analyzers.

This is a continuation of application Ser. No. 07/747,179, filed Aug.19, 1991, now U.S. Pat. No. 5,413,732.

BACKGROUND OF THE INVENTION

1. Field of Invention

This invention relates to novel reagent compositions and to methods fortheir preparation. In particular, it relates to novel lyophilizedreagent spheres and diluents useful in the analysis of biologicalsamples.

In preparing reagents for convenient and efficient testing of clinicalbiological samples, it is frequently important to obtain dry chemicalblends in uniform, discrete amounts. These reagents must be efficientlyand economically prepared in small precisely measured quantities.Reagents comprising organic materials, however, tend to spoil or degradeon storage, thus creating quality control problems. Thus, reagents aretypically provided in dried form to increase stability. Currenttechnology for producing dry chemical blends involves procedures such asdry blending, spray drying, or fluid bed drying. All three of theseprocedures, however, have limitations that make them costly, inefficientor difficult to carry out.

In dry blending technology, it is difficult to obtain homogeneous blendsof chemicals that have different densities. Moreover, homogeneity isparticularly difficult to achieve when very small amounts of ingredientsare mixed with large amounts of others. Once made homogeneous, it isextremely difficult to reproducibly (within 1 percent) dispense smallamounts (less than about 10 mg) of the blended chemicals.

Spray drying technology provides more homogenous blends of chemicalsbecause the reagents are first dissolved in liquid. Using spray drying,however, it is difficult and costly to obtain precisely sized amounts ofblended chemicals. As generally practiced, this process yields particleswith size distributions having coefficients of variation greater than 20percent. The resulting particles have to be reprocessed (usuallyagglomerated) to obtain uniform particle sizes. After agglomeration, theparticles are generally less soluble than the original spray driedparticles. Moreover, these procedures typically use fluorocarboncryogenic solutions which are hazardous to the environment.

Fluid bed technology relies upon spraying a liquid reagent blend onto aparticle and drying the liquid to obtain a particle coated with theblended reagents. Using this procedure, it is difficult to obtainuniformly sized particles and to produce a uniform coating.

Of particular interest to the present invention are reagents useful inanalyzing biological samples, such as blood plasma or serum, incentrifugal analyzers. The rotors used in such analyzers measure volumesof the sample to be tested, mix the sample with an appropriate diluentand separate fluid from cellular components. The rotors also provide aplurality of separate test wells containing chemical reagents in whichdiscrete volumes are optically tested.

Analysis of biological samples in the test wells of centrifugal rotorsimpose a number of requirements on the reagents used for analysis. Inparticular, because the analysis is typically highly automated, speed ofanalysis is at a premium. In addition, many clinical diagnostic analysesrequire that measurements be made within a short time after the sampleis added to the reagent. Thus, the dried reagent preparations mustdissolve quickly in the sample solution. In addition, rapid rehydrationof the reagents can cause bubble formation, which adversely affectsresults by interfering with optical measurement.

In centrifugal analyzers, the sample is typically mixed with a diluentbefore analysis. It is not possible to directly measure the amount ofdiluent added while the diluted sample is in the rotor. Obviously,improperly diluted samples will produce erroneous results. Thus,convenient methods for determining amount of dilution of the sample insitu are required. In addition, if the sample to be diluted comprisescells, the diluent must contain isotonic concentrations of compounds toprevent osmotic shock to the cells. Such compounds, however, must notenhance or inhibit any of the analyses. Many isotonic solutions aredisclosed in the prior art, including saline, glucose, or phosphatebuffered saline solutions. None of these solutions are suitable becausethey can affect results, because they provide additional bufferingcapacity to the solution or because they add chemicals which are thesame as the analytes of interest.

The prior art thus lacks reagent compositions which avoid the aboveproblems in centrifugal analyzers. In particular, the prior art lackseconomical and reliable reagent preparations which dissolve quickly insample solutions and avoid bubble formation. Moreover, currentlyavailable diluents are not suitable because dilution cannot be easilymeasured and they can alter the results of the analysis. The presentapplication addresses these and related problems.

2. Description of Background Art

U.S. Pat. Nos. 3,721,725 and 3,932,943 relate to methods for producinglyophilized reagents comprising spraying a solution containing thereagents into a moving bath of fluorocarbon refrigerants andlyophilizing the resultant frozen droplets. 4,848,094 discloses methodsfor the generation of essentially spherical frozen droplets and improvedmethods for removing frozen droplets from a cryogenic liquid. 4,655,047describes methods for freezing drops of relatively thick liquids bydropping them from a small height into a cryogenic material. 3,819,488provides stable lyophilized diagnostic compositions for determiningglutamic oxalic transaminase and glutamic pyruvic transaminaseactivities. 4,588,696 relates to preparation of tablets used in testingfor formaldehyde and/or glutaraldehyde. 4,295,280, 4,351,158, and4,712,310 all relate to methods for preparing homogenous preparationscomprising compounds which are incompatible. 4,820,627 discloses afluidized bed process for preparing particles suitable for tabletinginto diagnostic reagents. 4,115,537 relates to diagnostic tabletscontaining ion exchange resins. 4,755,461 is directed to tableted bloodplasma compositions. 4,678,812 and 4,762,857 both relate to diagnostictablets comprising trehalose as an excipient and stabilizer. The use ofTRITON®X-100 is also disclosed. 4,716,119 discloses the addition oftetramethylammonium acetate to blood serum. Romanian Patent Appln. No.85,155 relates to enzymatic alkaline phosphotase reagent tabletscomprising p-nitrophenyl phosphate. Driscoll et al., Clin. Chem.,29:1609-1615 (1983) discloses an instrument/reagent system comprisingtableted reagents for performing photometric assays.

SUMMARY OF THE INVENTION

The present invention is directed to compositions for analyzingbiological samples. In particular, it is directed to reagent spherescomprising reagents for analyzing the sample and methods for producingthe reagent spheres. The reagent spheres of the present invention arecapable of quickly and completely dissolving in a solution, typically inless than about ten seconds. The reagent spheres have a diameter betweenabout 1.7 mm and about 2.3 mm and have a coefficient of weight variationless than about 3%. The reagent spheres comprise, in addition to thereagents necessary for analysis of the biological sample, a surfactantat a concentration sufficient to inhibit bubble formation when thesphere dissolves and a filler in a concentration sufficient tofacilitate formation of a chemical lattice capable of conducting waterinto the reagent sphere.

The surfactant is typically a non-ionic detergent such as octoxynol 9(TRITON®X-100) or polyoxyethylene 9 lauryl ether. Concentration of thesurfactant in the reagent sphere is typically adjusted such that theconcentration in the reconstituted reagent is between about 0.08 g andabout 3.1 g per 100 ml.

Fillers suitable for use in the present invention arepolyethyleneglycol, myo-inositol, polyvinylpyrrolidone, bovine serumalbumin, dextran, mannitol, sodium cholate or a combination thereof. Thefiller compounds are typically present in concentration between about10% and about 50% by dry weight. The chemical lattice formed by thefiller compounds allows the reagent sphere to quickly and completelydissolve in a sample solution or diluent.

Reagent spheres are formed by preparing (an aqueous solution) of theappropriate reagent(s), dispensing uniform, precisely measured drops ofthe aqueous solution into a cryogenic liquid, and lyophilizing thefrozen drops. The cryogenic liquid is typically liquid nitrogen which isnot agitated.

Also provided are diluents suitable for mixing with a biological samplebefore optically analyzing the sample. Diluents of the present inventioncomprise an isotonic concentration of a compound which does notinterfere with the analysis of the sample. In particular, the preferredcompound will have substantially no buffer capacity at the pH of theparticular assay. Typical compounds for this use includetetramethylammonium acetate at a concentration between about 120 mM andabout 150 mM and inositol between about 20 and about 30 g/L. The diluentmay also comprise a photometrically-detectable marker compound fordetermining the dilution of the biological sample. Typical markercompounds include dyes such as1,1',3,3,3',3'-hexamethylindotricarbocyanine iodide,1,1'-bis(sulfoalkyl)-3,3,3',3'-tetramethylindotricarbocyanine salts,enzyme substrates (such as lactate and p-nitrophenylphosphate) andenzymes (such as D-lactate dehydrogenase and microbialglucose-6-phosphate dehydrogenase).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides compositions for analyzing biologicalsamples in, for example, centrifugal rotors and analyzers which allowrapid and economical analysis of blood samples. Lyophilized reagentspheres are provided that comprise a chemical lattice to facilitaterapid and complete dissolution of the spheres in an aqueous solution.They also comprise a surfactant at a concentration sufficient to inhibitbubble formation as the reagent spheres dissolve. The reagent spheresmay be used in combination with diluent solutions comprising isotonicconcentrations of compounds having substantially no effect on theassays. In addition, marker compounds are used to quickly and easilydetermine dilution of the sample in situ.

The reagent spheres and diluents of the present invention are suitablefor use in centrifugal analyzers for optically analyzing biologicalfluids, in particular blood plasma or serum. Centrifugal rotors used insuch analyzers typically comprise means for mixing the blood with anappropriate diluent and separating plasma from cellular material. Therotors also provide for distribution of the diluted plasma into aplurality of cuvettes within the rotor so that different opticalanalytic procedures may be performed without having to transfer aliquotsof the fluid from the apparatus. One or more reagent spheres comprisingthe reagents necessary for a desired assay are provided in each cuvette.

The rotors and methods described in the following copending applicationsare preferably used: U.S. Ser. Nos. 532,524, filed Jun. 24, 1990, andthree applications filed Apr. 1, 1990 (U.S. Ser. Nos. 678,823, 678,824,and 07/678,762). The entire disclosure of these applications areincorporated herein by reference. The above applications disclosecentrifugal rotors for separating plasma from whole blood that include aplurality of internal chambers and passages for combining blood plasmaor serum with one or more reagents and distributing the plasma or serumto a plurality of individual test wells. The chambers and passagesnecessary for separating the whole blood into plasma are locatedradially outward from metering chambers that deliver precisely measuredvolumes of blood and/or diluent to a separation chamber. The separationchamber includes a radially-outward cell trap. Spinning of the rotorcauses the cellular components of the whole blood to be sequestered inthe cell trap. The separated plasma is then delivered to a plurality oftest wells or cuvettes. The above separation and aliquoting stepstypically occur as a result of centrifugal force generated by thespinning rotor.

The compositions of the present invention in combination with the rotorsdescribed above are particularly suitable for analyzing blood plasma ordiluted blood plasma. They are also useful with a wide variety of otherbiological fluids, such as urine, sputum, semen, saliva, ocular lensfluid, cerebral fluid, spinal fluid, amniotic fluid, and tissue culturemedia, as well as food and industrial chemicals, and the like.

The compositions of the present invention are particularly suitable forperforming a wide variety of analytic procedures which are beneficiallyor necessarily performed on blood plasma or diluted plasma. The analyticprocedures will generally require that the blood plasma be combined withone or more reagents so that some optically detectable change occurs inthe plasma which may be related to measurement of a particular componentor characteristic of the plasma. Preferably, the plasma will undergo areaction or other change which results in a change in color,fluorescence, luminescence, or the like, which may be measured byconventional spectrophotometers, fluorometers, light detectors, etc. Insome cases, immunoassays and other specific binding assays may beperformed in the test wells. Generally, however, such assay proceduresmust be homogeneous and do not require a separation step. In othercases, it will be possible to accommodate heterogeneous assay systems byproviding a means to separate blood plasma from the test wells after animmunological reaction step has occurred.

Conventional blood assays which may be performed include glucose,lactate dehydrogenase, serum glutamic-oxaloacetic transaminase (SGOT),serum glutamic-pyruvic transaminase (SGPT), blood urea (nitrogen) (BUN),total protein, alkalinity, alkaline phosphatase, c-reactive proteinbilirubin, calcium, chloride, sodium, potassium, magnesium, and thelike. This list is not exhaustive and is intended merely as beingexemplary of the assays which may be performed using the apparatus andmethod of the present invention. Usually, these tests will require thatthe blood plasma be combined with one or more reagents which result in avisually detectable, usually photometrically detectable, change in theplasma.

Thus, the reagent spheres of the present invention are prepared fromreagents suitable for any of the above analytical assays. Typically, anaqueous solution comprising the reagents is prepared. To ensure uniformcomposition of the reagent spheres, the solution must be homogeneous andall constituents must be fully dissolved or in suspension. Individualdrops of the solution are then dispensed into a cryogenic liquid,preferably liquid nitrogen. A cryogenic liquid as used herein refers toa liquified gas having a normal boiling point below about -75° C.,preferably below about -150° C.

The frozen masses are then lyophilized to produce the reagent spheres.The reagent spheres typically comprise less than about 6% residualmoisture, preferably less than about 3%. Lyophilization is carried outaccording to standard procedures known in the art. Typically, the frozendrops are lyophilized for about 4 hours to about 24 hours at about 50 toabout 450 mTorr, preferably, about 6 hours at about 200 mTorr.

The drops are uniform and precisely measured so that the resulting driedreagent spheres have uniform mass. When the drops are uniform andprecisely measured, the imprecision of the mass (coefficient of weightvariation) of the reagent spheres prepared from the drops is less thanabout 3%, and preferably between about 0.3% and about 2.5%. To furtherdecrease the coefficient of weight variation, the aqueous solution ispreferably degassed using a vacuum pump or vacuum line before the dropsof solution are dispensed.

To obtain values for coefficient of weight variation, known quantitiesof reagent spheres are weighed. The coefficient of variation (C.V.) isthen determined as follows: ##EQU1## wherein J= ##EQU2## x=weight of onesphere x=mean (for "n" sphere)=Σx/n

The uniformity of the reagent spheres produced by this method obviatesthe need for an additional tableting step to obtain uniform size. Thedrops can be dispensed by any of a number of means which provide thenecessary precision. Typically, an IVEK model AAA pump (N. Springfield,Vt.) is used. The solution is usually dispensed in discrete drops havinga volume between about 2.5 μl and about 4.0 μl. The exact volume of thedrops will depend upon the particular application. For instance, inpreparing reagent spheres for total protein determinations, 2.96 μldrops are typically used, for C-reactive protein and alkalinephosphatase determinations, 2.67 μl are used. Volumes appropriate forother tests are as follows: SGOT, 4.0 μl; potassium, 4.0 μl; creatinine,4.0 μl; bilirubin, 2.667 μl; amylase, 2.667 μl; cholesterol, 2.667 μl;uric acid, 3.478 μl; and glucose, 2.065 μl.

The reagent spheres of the present invention dissolve quickly in anaqueous sample solution, or diluent. A sample solution of the presentinvention may be a diluted or undiluted biological sample. The reagentspheres typically dissolve in less than about 30 seconds, preferablyless than about 10 seconds. The rapidity of dissolution gives theimpression that the reagent sphere "explodes" and distributes thedissolving chemicals throughout the reconstituting volume. Rapiddissolution of the spheres is facilitated by a chemical latticestructure which quickly conducts water into the reagent sphere. To formthe chemical lattice, fillers are included in the aqueous solution usedto produce the spheres. As the reagent spheres are lyophilized, thesemolecules facilitate formation of a network of open spaces or a chemicallattice in the spheres. The filler components of the reagent spheres aretypically polymeric compounds, such as bovine serum albumin,polyethylene glycol, dextran, FICOLL® (polysucrose) (Pharmacia LKBBiotechnology, Inc., Piscataway, N.J.), or polyvinylpyrrolidone. Inaddition, emulsifiers such as sodium cholate and the like are useful asfillers. Monosaccharides and their derivatives, such as mannitol or thepolyalcohol, myo-inositol, can also be used. Depending upon the assay,the fillers can be used individually or in combination with one or moreof the other filler components.

In addition to fillers, the reagent spheres of the present inventionalso comprise one or more surfactants at concentrations sufficient toinhibit bubble formation when the spheres are rapidly rehydrated. Asdescribed above, bubbles are detrimental to the assays because theyinterfere with optical measurements. If the reagent spheres comprisesurfactants at the appropriate concentrations, however, such problemsare avoided. Suitable surfactants include non-ionic detergents such aspolyoxyethylene 9 lauryl ether, octoxynol 9, SYNTHRAPOL®(polyoxoethylene alkyl alcohol) NP-90, TRYCOL® (polyoxoethylene tridecylalcohol 6735) 5941, Trycol® 6735 and the like. Ionic detergents such asGAFAC® (polyoxothylene nonylphenyl ether phosphate sodium salt) 560,sodium dodecyl sulfate and the like are also suitable. Typically, thesurfactants are present in the reconstituted reagent spheres at aconcentration between about 0.08 g and about 3.1 g per 100 ml. Thesurfactant concentration used will depend upon the particular reagentsused in the assay.

The fillers and surfactants used in a particular reagent spherepreparation are preferably selected so as to minimize interference withthe assay. Optimization of the these components is facilitated by Table1 which provides information regarding desired characteristics offillers and surfactants suitable for use with reagents used in a varietyof assays. In addition, the Example section below provides the preciseconcentrations of filler and surfactant components which have been foundto be particularly useful in the exemplified assays.

In order to provide reagent spheres of the correct size in a test well,the components are typically concentrated in the reagent sphere. Uponrehydration with a predetermined volume of sample, the reagents andother components are present in the correct concentration. For instance,the components of the reagent spheres for alkaline phosphatedeterminations are typically at about 6× concentration and total proteinreagents are at about 2.7× concentration. The ideal concentration forthe reagents for particular assay can be easily determined, dependingupon size of the test well, sample volume, and the like.

                                      TABLE 1                                     __________________________________________________________________________    (g/100 ml)                                                                           fillers                        surfactants                                    PEG                                                                              PEG                                                                              PEG     bovine           Mega                                                                              n-Octyl                                                                            Triton      cholic                    3400                                                                             8000                                                                             20M                                                                              Dextran                                                                            albumin                                                                            PVP                                                                              inositol                                                                          mannitol                                                                           8   glucoside                                                                          X-100                                                                             Trycol                                                                            Thesit                                                                            acid               __________________________________________________________________________    ALP A        5.40         0.10                                                                             1.0               0.08                           ALP B        5.40         0.10                                                                             1.0               0.08                           Amylase   4.00               2.0               0.30                           AST       2.50  2.50 2.50                      0.40                           BUN       4.00                                 0.30                           Cholesterol                                                                             0.87       3.70                                                     (BMD)                                                                         CRP          8.40                              0.30                           Creatinine                                     0.21        2.0                Test                                                                          Creatintine                                    0.25        2.0                Blank                                                                         Glucose   1.80       2.10                      0.30                           Plasma 6.0   1.00    2.00    1.0               0.38                                                                              2.10    10.0               Dilution                                                                      Rotor Q.C.A                                                                          8.0   3.00                              0.50                           Rotor Q.C.B                                                                          5.0   2.00            1.0               0.50                           Sample    9.60                                 0.80                           Blanking                                                                      Temperature     1.00      0.10   10.00                                                                              0.20                                                                              0.20                                Bilirubin       2.00         1.0 6.00                                         Enzyme                                                                        Bilirubin 8.00                                                                Buffer                                                                        Total Protein                                                                        5.00                                                                             4.00                                                                             0.50                                      0.143                                                                             0.5                Triglycerides                                                                           1.80  3.60 3.60                      0.15                                                                              0.30    0.1                Blank                                                                         Triglycerides                                                                           1.80  3.60 3.60                      0.15                                                                              0.30    0.1                Test                                                                          Uric Acid 4.00                                 0.24                           __________________________________________________________________________

As discussed above, diluents are typically used in assays of biologicalsamples. Diluents for use with samples which contain intact cells, suchas whole blood, must comprise isotonic concentrations of compounds toprotect the cells from osmotic shock. The presence of the isotoniccompounds in the diluent, however, must have substantially no effect onthe results of the assay. An isotonic compound has substantially noeffect on an assay if its presence leads to less than about a 5% changein the results of a quantitative assay, preferably less than about 2.5%,and most preferably less than about 1%. In particular, additional buffercapacity provided by the isotonic compounds should be minimized. Thepresent invention provides improved diluents comprising isotonicconcentrations of compounds which do not interfere with the assays. Thisis accomplished by, for instance, selecting salts of weak acids withpKa's outside the pH range of the particular assay. A preferred salt ofa weak acid for this purpose is tetramethylammonium acetate at aconcentration of about 120 to about 150 mM. Other suitable compoundsinclude myo-inositol, a polyalcohol which has no buffering capacity, atconcentrations from about 2% to about 3%.

The diluents of the present invention may also comprise marker compoundswhich allow the user to quickly and easily determine dilution in situ.The marker compounds of the present invention are typicallyphotometrically detectable compounds which are added in predetermined ormeasurable amounts to the diluent. After mixing the diluent with thesample, the concentration of the marker is photometrically determined.This can be done by, for instance, comparing the absorbance of thediluted sample at the appropriate wavelength to standard solutions ofknown concentration. The ratio of the concentrations of the markerbefore and after mixing can then be used to determine the amount ofdilution of the sample.

Various photometrically detectable marker compounds can be used.Compounds which can be used in a photometrically detectable colorreaction can also be used. Ideally, the marker compound does not absorbat any of the wavelengths used in any of the analyses or causeinterference with any subsequent assays performed on the sample. Dyessuch as 1,1'3 3 3'3'-hexamethylindotricarbocyanine iodide or1,1'-bis(sulfoalkyl)-3,3,3',3'-tetramethylindotricarbocyanine salts aretypically used. Suitable marker compounds which are converted tophotometrically detectable compounds include enzyme substrates notnormally present in the sample, such as p-nitrophenyl phosphate orD-lactate. The compound p-nitrophenylphosphate is a substrate foralkaline phosphatase and yields a colored p-nitrophenol reactionproduct. D-lactate is a substrate for D-lactate dehydrogenase and whenused with NAD produces the colored NADH reaction product. Other possiblemarkers suitable for use in the diluent include enzymes themselves ifused with substrates either present in the plasma or in the reactionchambers that produce color. The enzymes should not normally be presentin the sample. For samples of human origin, typical enzymes includemicrobial glucose-6-phosphate dehydrogenase and D-lactate dehydrogenase.Obviously, the marker compound is preferably selected so as to minimizeinterference with any subsequent assays performed on the sample. Incases where the marker compound is unstable and long term storage of thediluent is not practical, the marker or its precursor can be held in thedry state and solubilized near or at the time of its use. Such anexample is 1,1'-bis(sulfoalkyl)-3,3,3'3'-tetramethylindotricarbocyaninesalts which after solubilization in aqueous solutions aggregates. Toprevent these problems, the indocyanine dyes and other unstable dyes aretypically stored in a dry form, applied to a solid surface. The solidsurface may be, for instance, the wall of a passage, capillary orchamber in the analytical rotor or an inert carrier, such as apolystyrene ball. The surface comprising the adsorbed dye may be placedin any passage or chamber in the analytical rotor, for instance in apassage between the diluent chamber and the mixing chamber, or in themixing chamber. A suitable isotonic diluent solution then dissolves thedye off the surface at the time of its use. The aqueous diluent isselected according to the particular dye used. For indocyanine dyes,2.5% myo-inositol is suitable.

The following examples show preparation of reagent spheres forparticular assays. These examples are provided by way of illustrationand not by way of limitation.

EXAMPLE 1 Preparation of Reagent Spheres for Total Protein Determination

The following solution was prepared by accurately weighing anddissolving the following chemicals in a container of about 800 ml ofdeionized or distilled water:

    ______________________________________                                        sodium potassium tartrate                                                                              37.80  g                                             sodium hydroxide pellets 28.20  g                                             cupric sulfate           12.00  g                                             potassium iodide         12.90  g                                             sodium carbonate         3.85   g                                             sodium cholate           5.00   g                                             polyoxyethylene 9 lauryl ether                                                                         1.43   g                                             polyethylene glycol (FW 3400)                                                                          50.00  g                                             polyethylene glycol (FW 8000)                                                                          40.00  g                                             polyethylene glycol (FW 20,000)                                                                        5.00   g                                             ______________________________________                                    

It is best to completely dissolve each chemical before adding the nextchemical. After the last chemical dissolved, the solution volume wasadjusted to 1.0 liter with deionized or distilled water. The solutionwas filtered through a stack of media that terminated in 0.45 micronporosity. The solution was then degassed using a vacuum pump. The abovesolution when diluted 37 ml plus 63 ml with water is used to assay Totalprotein concentration in various clinical samples such as serum orplasma. The sodium carbonate is added as a stabilizer, andpolyoxyethylene 9 lauryl ether is added for controlling bubbles duringdissolution. Sodium cholate and the various polyethylene glycols areadded as fillers to facilitate formation of a chemical lattice duringsubsequent freeze drying.

The solution was dispensed by an IVEK model AAA pump in discrete 2.96microliter drops at a rate of 1 to 2 drops-per-second. The discreteamounts of fluid drop through air, form spheres and land on the surfaceof liquid nitrogen. The surface of the nitrogen does not need to beagitated. After freezing the spheres were dried in Virtis freeze dryer(model no. 12EL console) (Gardener, N.Y.) until their residual moistureswere less than 11% of the total remaining mass. A freeze dried reagentsphere prepared according to the above method can be reconstituted with8 microliters of a mixture of water or diluent (14 parts) and humanserum (1 part). The resulting change-in absorbance at 550 nm minus theabsorbance of a reagent sphere reconstituted with 8 microliters of wateror diluent and minus the absorbance of the human serum sample diluted inthe same ratio with water plus polyethylene lauryl ether and sodiumcholate is proportional to the amount of total protein in the sample.

The imprecision (coefficient of variation) among the 1.78 millimeterdiameter spheres is:

    ______________________________________                                        dispensed frozen spheres                                                                           1.5% at 3.7 mg                                           freeze dried spheres 2.5% at 0.6 mg                                           ______________________________________                                    

Each reagent sphere dissolves in 8 microliters of water or diluentwithin 5 seconds in a centrifugal analyzer.

EXAMPLE 2 Preparation of Reagent Spheres for C-Reactive ProteinDetermination

The following solution was prepared by accurately measuring weighing anddissolving the following chemicals in a container of about 200 mls ofdeionized or distilled water:

    ______________________________________                                        C-reactive protein antibody                                                                           0.56   liters                                         Sodium chloride         25.50  g                                              HEPES                   71.50  g                                              Triton ® X-100      3.00   g                                              polyethylene glycol (FW 20,000)                                                                       84.00  g                                              ______________________________________                                    

It is best to completely dissolve each chemical before adding the nextchemical. After the last chemical dissolved, the pH was adjusted to 7.4with dilute sodium hydroxide and the solution volume was adjusted to 1.0liter with deionized or distilled water. The solution was filteredthrough a stack of media that terminated in 0.2 micron porosity. Thesolution was then degassed.

The above solution when diluted 33 ml plus 67 ml with water or diluentis used to assay C-reactive protein in various clinical samples such asserum or plasma. The sodium chloride is added as a stabilizer andTriton®X-100 is added for controlling bubbles during dissolution.Polyethylene glycol is added to facilitate the development of turbidityin the analytic reaction and as filler to facilitate formation of achemical lattice during subsequent freeze drying.

The solution was dispensed by an IVEK model AAA pump in discrete 2.67microliter drops at a rate of 1 to 2 drops-per-second. The discreteamounts of fluid drop through air, form spheres and land on the surfaceof liquid nitrogen. The surface of the nitrogen does not need to beagitated. After freezing, the spheres were dried in a Virtis freezedryer (model no. 12EL console) until their residual moistures were lessthan 6% of the total remaining mass.

A freeze dried reagent sphere prepared according to the above method canbe reconstituted with 8 microliters of a mixture of water or diluent (14parts) and human serum (1 part). The resulting change in absorbance at340 nm minus the absorbance of a reagent sphere reconstituted with 8microliters of water or diluent and minus the absorbance of the humanserum sample diluted in the same ratio with water plus Tritone®X 100 isproportional to the amount of C-reactive protein in the sample.

The imprecision (coefficient of variation) among the 1.72 millimeterdiameter spheres is:

    ______________________________________                                        dispensed frozen spheres                                                                           1.7% at 2.9 mg                                           freeze dried spheres 1.8% at 0.5 mg                                           ______________________________________                                    

Each reagent sphere dissolves in 8 microliters of water or diluentwithin 3 seconds in a centrifugal analyzer.

EXAMPLE 3 Preparation of Reagent Spheres for Alkaline Phosphatase (ALP)Determination

The following solutions were prepared. ALP part A: The followingchemicals were accurately measured, weighed, and dissolved in acontainer of about 800 mls of deionized or distilled water:

    ______________________________________                                        Tris(hydroxymethyl)aminomethane-HCL                                                                     10.2   g                                            HEDTA                     2.1    g                                            magnesium chloride hexahydrate                                                                          2.6    g                                            zinc sulfate heptahydrate 1.7    g                                            4-nitrophenylphosphate    35.6   g                                            polyethylene glycol (FW 20,000)                                                                         54.0   g                                            myo-inositol              10.0   g                                            Triton ® X-100        0.8    g                                            glycerol                  6.0    g                                            polyvinylpyrrolidone (FW 30,000)                                                                        1.0    g                                            ______________________________________                                    

It is best to completely dissolve each chemical before adding the nextchemical. After the last chemical dissolved, the pH was adjusted to 6.8with dilute 2-amino-2-methyl-1-propanol and the solution volume wasadjusted to 1.0 liter with deionized or distilled water. The solutionwas filtered through a stack of media that terminated in 0.2 micronporosity. The solution was then degassed.

ALP part B: The following chemicals were accurately measured, weighed,and dissolved in a container of about 800 mls of deionized or distilledwater:

    ______________________________________                                        Tris(hydroxymethyl)aminomethane-HCL                                                                     10.2   g                                            Tris(hydroxymethyl)aminomethane                                                                         166.0  g                                            HEDTA                     2.1    g                                            polyethylene glycol (FW 20,000)                                                                         54.0   g                                            myo-inositol              10.0   g                                            Triton ® X-100        0.8    g                                            2-amino-2-methyl-1-propanol                                                                             53.4   g                                            polyvinylpyrrolidone (FW 30,000)                                                                        1.0    g                                            ______________________________________                                    

It is best to completely dissolve each chemical before adding the nextchemical. After the last chemical dissolved, the pH was adjusted to 10.3with dilute 2-amino-2-methyl-1-propanol and the solution volume wasbrought to 1.0 liter with deionized or distilled water. The solution wasfiltered through a stack of media that terminated in 0.2 micronporosity. The solution was then degassed.

The above solutions when combined in equal volumes of 16.7 ml each and67 ml of water or diluent are used to assay alkaline phosphatase invarious clinical samples such as serum or plasma. The glycerol is addedas a stabilizer, Triton®X100 is added for controlling bubbles duringdissolution. Polyethylene glycol, myo-inositol, and polyvinylpyrrolidoneare added as fillers to facilitate formation of a chemical latticeduring subsequent freeze drying.

The solutions were dispensed separately by an IVEK model AAA pump indiscrete 2.67 microliter drops at a rate of 1 to 2 drops-per-second. Thediscrete amounts of fluid drop through air, form spheres and land on thesurface of liquid nitrogen. The surface of the nitrogen does not need tobe agitated. After freezing the spheres were dried in a Virtis freezedryer (model no. 12EL console) until their residual moistures were lessthan 6% of the total remaining mass.

One of each, ALP A and ALP B, freeze dried reagent spheres can bereconstituted with 16 microliters of a mixture of water or diluent (14parts) and human serum (1 part). The resulting rate of change inabsorbance at 405 nm is proportional to the amount of alkalinephosphatase in the sample.

The imprecision (coefficient of variation) among the 1.72 millimeterdiameter spheres is:

    ______________________________________                                                     ALP A      ALP B                                                 ______________________________________                                        dispensed frozen spheres                                                                     0.4% at 2.8 mg                                                                             0.7% at 2.9 mg                                    freeze dried spheres                                                                         1.5% at 0.5 mg                                                                             2.2% at 0.7 mg                                    ______________________________________                                    

The two reagent spheres dissolve in 16 microliters of water or diluentwithin 10 seconds in a centrifugal analyzer. The active constituents inthis assay are separated to improve reagent stability. One of each ofthe spheres is placed in the same chamber for the ALP assay.

EXAMPLE 4 Preparation of Freeze-Dried Concentrated Potassium ReagentContaining Macrocyclic Ionophore Trinitroanilino Cryptahemispherand[2.2] for Potassium Determination

The active trinitroanilino cryptahemispherand [2.2] and surfactants(BRIJ® surfactants) were isolated from CHROMOLYTE™ Potassium Reagent(Technicon Instruments Corp., Tarrytown, N.Y. 10961) using Wide-PoreButyl, 40μM chromatographic medium (J. T. Baker Inc., Phillipsburg, N.Y.08865) as follows:

25 g of Wide-Pore Butyl, 40μM chromatographic medium were suspended in360 ml of degassed isopropanol and then 360 ml of degassed deionizedwater was added. About 80% of the liquid was decanted. An additional 360ml portion of degassed deionized water was added and the slurry in theflask was sonicated for two minutes, followed by two minutes of vacuumdegassing. The suspended chromatographic medium was poured into anappropriately sized chromatographic column to form a 3-10 cm highpacking bed. The packing was equilibrated by passing 250 ml of degasseddeionized water through the column.

Five liters of ChromoLyte™ Potassium Reagent were applied to the column.The colored trinitroanilino cryptahemispherand [2.2] and the surfactantswere adsorbed on the top of the column. The nonadsorbed triethanolaminebuffer, also containing 3% of 2-(2-ethoxyethoxy)-ethanol (EEE) andstabilizers, was collected and saved for later use. The trinitroanilinocryptahemispherand [2.2] and the surfactants were eluted from the columnwith a mixture of previously degassed isopropanol (98%) and EEE (2%).The isopropanol was removed from the eluate using an evacuated rotaryevaporator at room temperature to yield an oily, dark brown concentrate.

The previously collected buffer fraction was concentrated twofold usingan evacuated rotary evaporator at 35°-40° C. The concentratedtrinitroanilino cryptahemispherand [2.2], the surfactants and theremaining EEE were dissolved in 400 ml of the twofold concentratedbuffer solution.

The following materials were measured, added and dissolved in the abovesolution:

    ______________________________________                                        polyethylene glycol (FW 3400)                                                                         40     g                                              isopropanol             5.0    ml                                             polyvinylpyrrolidone K-29-32                                                                          0.50   g                                              ______________________________________                                    

The pH of the solution was measured using an electrode pair with acalomel reference electrode to verify that the pH was less than 0.05 pHunit different from the pH of the starting CHROMOLYTE™ PotassiumReagent. If necessary, pH adjustment was made with a 20% triethanolaminesolution or with a 20% triethanolamine HCl solution. Finally, the volumewas adjusted to 500 ml with the twofold concentrated buffer solution.The reagent was filtered through a stock of media that terminated in 0.2micron porosity. The preferred concentration of2-(2-ethoxyethoxy)-ethanol is between about 3% and about 4.8%, and thatof the polyethoxylauryl ether is between about 0.5 and about 1.0%.

The above solution when diluted 50 ml plus 50 ml with water or diluentis used to assay potassium in various clinical samples such as serum orplasma. The level of 2-(2-ethoxyethoxy)-ethanol is necessary to insureuniform freezing of the reagent and to aid in rapid resolubilizationafter freeze-drying. The isopropanol aids in creating the correctcrystal structure during the freezing process so that the rehydration isfacilitated. The BRIJ® surfactant (e.g., BRIJ® -35 or -58) aids inrehydration and in bubble inhibition. The polyethylene glycol is addedto facilitate formation of a chemical lattice during subsequent freezedrying.

The solution was dispensed by an IVEK model AAA pump in discrete 4.0microliter drops at a rate of 1 to 2 drops-per-second. The discreteamounts of fluid drop through air, form spheres were dried in a Virtisfreezer dryer (model no. 12 EL console) until their residual moistureswere less than 6% of the total remaining mass. A freeze dried reagentsphere prepared according to the above method can be reconstituted with8 microliters of a mixture of water or diluent (14 parts) and humanserum (1 part). The resulting change in absorbance at 500 nm minus theabsorbance of a reagent sphere reconstituted with 8 microliters of wateror diluent and minus the absorbance of the human serum sample diluted inthe same ratio with water plus BRIJ® surfactant is proportional to theamount of potassium in the sample.

The imprecision (coefficient of variation) among the 1.97 millimeterdiameter spheres is:

    ______________________________________                                        dispersed frozen spheres                                                                           1.5% at 2.6 mg                                           freeze dried spheres 1.6% at 0.5 mg                                           ______________________________________                                    

Each reagent sphere dissolves in 8 microliters of water or diluentwithin 5 seconds.

The above examples illustrate preparation of particular reagent sphereswithin the scope of the present invention. The examples have beenprovided for the purposes of clarity and understanding the invention. Itwill be apparent, however, that certain changes and modifications may bepracticed within the scope of the appended claims.

What is claimed is:
 1. A method for forming reagent spheres comprisingreagents for analyzing a biological sample, the method comprising thesteps of:forming an aqueous solution of the reagents; dispensinguniform, precisely measured drops of the aqueous solution into acryogenic liquid, whereby the drops are frozen; lyophilizing the frozendrops, thereby forming the reagent spheres.
 2. A method of claim 1wherein the cryogenic liquid is unagitated.
 3. A method of claim 1wherein the reagent spheres have a mean diameter between about 1.5 mmand 2.3 mm.
 4. A method of claim 1 wherein the reagent spheres have acoefficient of weight variation less than about 3.0%.
 5. A method ofclaim 1 wherein the uniform, precisely measured drops have a volumebetween about 2.0 μl and about 6.5 μl.
 6. A method of claim 1 whereinthe aqueous solution is degassed before dispensing uniform, preciselymeasured drops.
 7. A method of claim 1 wherein the cryogenic liquid isliquid nitrogen.
 8. A method of claim 1 wherein the aqueous solutionfurther comprises a filler in a concentration sufficient to facilitateformation of a chemical lattice in the reagent spheres.
 9. A method ofclaim 8 wherein the filler is polyethylene glycol, myo-inositol,polyvinylpyrrolidone, dextran, sodium cholate, mannitol, bovine serumalbumin, or a combination thereof.
 10. A method of claim 1, wherein theaqueous solution further comprises a surfactant at a concentrationsufficient to inhibit bubble formation when the reagent spheres dissolvein a second solution.
 11. A method of claim 10 wherein the surfactantcomprises octoxynol 9 or polyoxyethlene 9 lauryl ether.
 12. A method ofclaim 1 wherein the solution is a sample solution, or a diluent.
 13. Areagent sphere made in accordance with the method of claim
 1. 14. Areagent sphere comprising reagents necessary for the analysis of abiological sample, a surfactant at a concentration sufficient to inhibitbubble formation when the sphere dissolves and a filler in aconcentration sufficient to faciliate formation of a chemical laticecapable of conducting the solution into the reagent sphere.
 15. Areagent sphere of claim 14 which completely dissolves in less than about20 μl of solution.
 16. A reagent sphere of claim 14 which completelydissolves in less than about 10 seconds in an aqueous solution.
 17. Areagent sphere of claim 14 having a diameter between about 1.5 mm and2.3 mm.
 18. A reagent sphere of claim 14 wherein the filler ispolyethylene glycol, myo-inositol, polyvinylpyrrolidone, dextran, sodiumcholate, mannitol, bovine serum albumin or a combination thereof.
 19. Areagent sphere of claim 14 wherein the concentration of the filler isbetween about 10% and about 50% by weight.
 20. A reagent sphere of claim14 wherein the surfactant comprises octoxynol 9, BRIJ®-35, BRIJ®-58, orpolyoxyethylene 9 lauryl ether.
 21. A reagent sphere of claim 14 whereinthe concentration of the surfactant is adjusted such that, aftercompletely dissolving, the concentration of the surfactant in the sampleis between about 0.08 g and about 3.1 g per 100 ml.
 22. A reagent sphereof claim 14 wherein the reagents are suitable for determination of totalprotein in a blood sample, the filler is polyethylene glycol and thesurfactant is polyoxyethylene 9 lauryl ether.
 23. A reagent sphere ofclaim 14 wherein the reagents are suitable for determination ofC-reactive protein in a blood sample, the filler is polyethylene glycoland the surfactant is octoxynol
 9. 24. A reagent sphere of claim 14wherein the reagents are suitable for determination of alkalinephosphatase activity in a blood sample, the filler comprisespolyethylene glycol, myo-inositol and polyvinylpyrrolidone and thesurfactant is octoxynol
 9. 25. A reagent sphere of claim 14 wherein thereagents comprise a chromogenic ionophoric cryptahemispherand.
 26. Areagent sphere of claim 25 further comprising a volatile organicsolvent, a surfactant, and a filler.
 27. A reagent sphere of claim 26wherein the organic solvent is isopropanol, the surfactant comprisesBRIJ®-35 or BRIJ®-58, and the filler is polyethylene glycol.
 28. Adiluent suitable for mixing with a biological sample before opticalanalysis of the sample, the diluent comprising an isotonic concentrationof a compound having substantially no buffer capacity in the analysisand a photometrically detectable marker compound for determining thedilution of the biological sample.
 29. A diluent of claim 28 wherein thecompound is tetramethylammonium acetate at a concentration between about120 mM and about 150 mM.
 30. A diluent of claim 28 wherein the compoundis myo-inositol at a concentration between about 2% and about 3%.
 31. Adiluent of claim 28 wherein the marker compound is an enzyme substrate.32. A diluent of claim 31 wherein the enzyme substrate is p-nitrophenylphosphate or D-lactate.
 33. A diluent of claim 28 wherein the markercompound is 1,1,3,3,3',3'-hexamethylindotricarbocyanine iodide or a1,1'-bis(sulfoalkyl)-3,3,3',3'-tetramethylindotricarbocyanine salt. 34.A diluent of claim 28 wherein the marker compound is an enzyme.
 35. Adiluent of claim 34 wherein the enzyme is glucose-6-phosphatedehydrogenase, or D-lactate dehydrogenase.
 36. A lyophilized compositioncapable of completely dissolving in a solution, the composition,comprising a chromogenic ionophoric cryptahemispherand in aconcentration sufficient to assay potassium in a biological sample. 37.A composition of claim 36 further comprising a surfactant at aconcentration sufficient to inhibit bubble formation when thecomposition dissolves in the solution.
 38. A composition of claim 36wherein the cryptahemispherand is trinitroanilino cryptahemispherand[2.2].
 39. A method for adding a marker compound to a solution in ananalytical rotor, the method comprising the steps of:applying the markercompound to a solid surface in the rotor; and contacting the solidsurface with the solution, thereby dissolving the marker compound in thesolution.
 40. A method of claim 39 wherein the marker compound is a1,1'-bis(sulfoalkyl)-3,3,3',3'-tetramethylindotricarbocyanine salt or1,1',3,3,3',3'-hexamethylindotricarbocyanine iodide.
 41. A method ofclaim 39 wherein the solid surface is a polystyrene ball placed in apassage or chamber in the analytical rotor.
 42. A method of claim 41wherein the step of contacting the solid surface with the solution iscarried out by spinning the rotor, thereby causing the solution to flowthrough the passage or into the chamber.
 43. A method of claim 42wherein the solution is a diluent.
 44. A method of claim 43 wherein thediluent comprises myo-inositol.
 45. A method for determining thedilution of a biological sample, the method comprising the stepsof:providing a diluent comprising a photometrically detectable markercompound; mixing the diluent with the biological sample, thereby forminga diluted sample; determining the amount of the marker in the diluent;and determining the amount of the marker in the diluted sample.
 46. Amethod of claim 45 wherein the step of mixing the diluent with thebiological sample is carried out in a centrifugal analyzer.
 47. A methodof claim 45 wherein the marker compound is p-nitrophenyl phosphate,D-lactate, 1,1',bis(sulfoalkyl)-3,3,3',3'-tetramethylindotricarbocyanine iodide,1,1',3,3,3',3'-hexamethylindotricarbocyanine iodide, glucose-phosphatedehydrogenase, or D-lactate dehydrogenase.
 48. A method for opticallyanalyzing a biological fluid, the method comprising,delivering apredetermined volume of the biological fluid to a test well containing areagent sphere comprising the reagents for the analysis of the sample,whereby the reagent sphere completely dissolves in the fluid; passing alight beam through the fluid in the test well; and detecting the lightbeam after it has passed through the fluid.
 49. A method of claim 48wherein the test well is in a centrifugal rotor and the step ofdelivering the predetermined volume of biological fluid is carried outby spinning the rotor.
 50. A method of claim 48 wherein the biologicalfluid comprises blood plasma or blood serum.
 51. A method of claim 48wherein the reagent sphere further comprise a surfactant at aconcentration sufficient to inhibit bubble formation when the spheredissolves and a filler in a concentration sufficient to facilitateformation of a chemical lattice capable of conducting the solution intothe reagent sphere.